Apparatus for two-step surface-enhanced raman spectroscopy

ABSTRACT

A kit of components is used in the detection, identification, analysis, and quantitation, by SERS, of a designated target analyte in a sample, comprising: packaging means normally containing a SER-active device component providing a support structure including a SER-active material, a collection component, a container component containing a liquid reagent comprised of a SER-active material, and a component for introducing the liquid reagent into the SER-active device. At least one of the SER-active materials is functionalized with an agent having the specific capability of binding the designated target analyte, and is accessible for the deposit thereon of liquid analyte samples. The SER-active device is constructed for receiving the liquid reagent and for enabling irradiation and collection of Raman-scattered radiation, by and from a Raman spectrometer, cooperatively generated by SER-active metals of the SER-active materials.

BACKGROUND OF THE INVENTION

The ability to measure trace quantities of biological materials in avariety of matrices is important to numerous fields. For example, themeasurement of a few Bacillus anthracis spores in air would beinvaluable to homeland security, the measurement Escherichia coli inwater or Salmonella enterica in food would be invaluable to publicsafety, and the ability to measure disease causing bacteria (e.g.methicillin resistant Staphylococcus aureus, MRSA), disease biomarkersor nucleotides in body fluids, such as blood, saliva, or urine, would beinvaluable to medical diagnosis. Three methods are widely used to detectsuch trace quantities of biological material, 1) culture growth ofbacteria or viruses that are detected by eye with or without the aid ofstaining and a microscope, 2) immunoassays in which the binding of anantigen to an antibody is detected or, more recently, 3) polymerasechain reactions in which primers are used to separate targetnucleotides, and polymerases are used to generate millions of copies ofthe nucleotides until they are detectable. The latter two methods oftenemploy fluorescent or radioactive labels for detection.

For most of the applications described above, effective analysisrequires speed, sensitivity, selectivity and, ideally, ease-of-use andportability to make at-site measurements. Rapid detection of B.anthracis spores in air is required for effective evacuation ofpotentially exposed personnel; rapid detection of a patient infectedwith MRSA is required to quarantine the patient and minimize spread ofthis disease; and rapid detection of E. coli in food is required tolimit distribution of spoiled food that may cause widespread illness.Extreme sensitivity is also required, as the Department of Defense hasestimated that the median lethal dose (LD₅₀) for weapons-gradeinhalation anthrax is as few as 2,500 B. anthracis spores (DefenseIntelligence Agency, Soviet biological warfare threat, DST-1610F-057-86,1986), while 10⁶ colony forming units (CFUs) of MRSA can cause aninfection (Freitas, R., “Microbivores: Artificial Mechanical PhagocytesUsing Digest and Discharge Protocol “, Journal of Evolution andTechnology, 14, 55-106, 2005), and 100 CFUs of E. coli on a foodproduct, considered an infectious dose, can become 1 billion CFUs duringa period of just eight hours of transport to market (E. coli CFUs doubleabout every 20 minutes, Irwin, P L et al., “Evidence for a bimodaldistribution of Escherichia coli doubling times below a thresholdinitial cell concentration”, BMC Microbiology, 10, 207; 2010).Specificity is also important, in that bacteria within a genus, species,subspecies, strain or serotype, such as B. cereus and S. aureus(non-methicillin resistant), can give false positive responses for B.anthracis and MRSA, members of the same genus and species respectively,using some analytical methods, potentially causing unnecessary anguishor treatment.

Unfortunately, none of the three methods described above can meet thelisted requirements: culture growth takes several days, PCR takesseveral hours, and immunoassays lack sensitivity. In addition, onlyimmunoassays are sufficiently portable for at-site measurements, such asat a military base or a food-processing center.

Recently, several researchers have proposed the use of surface-enhancedRaman spectroscopy (SERS) as the detection method for immunoassays. SERSinvolves the absorption of incident laser photons within nanoscale metalstructures, generating surface plasmons, which couple with nearbymolecules (the analyte) and thereby enhance the efficiency of Ramanscattering by six orders of magnitude or more (Jeanmaire D L, R P VanDuyne, “Surface Raman Spectroelectrochemistry”, J Electroanal Chem, 84,1-20, 1977; or Weaver M J, S Farquharson, M A Tadayyoni,“Surface-enhancement factors for Raman scattering at silver electrodes:Role of adsorbate-surface interactions and electrode structure”, J ChemPhys, 82, 4867-4874, 1985).

SERS has been shown to be capable of detecting trace quantities ofdipicolinic acid, a chemical prevalent in spores, such as B. anthracis,rapidly, and in an easy-to-use format (Farquharson et al., U.S. Pat. No.7,713,914). However, specificity was limited, and bacilli could not bedifferentiated from clostridia; and differentiation at the species level(e.g. B. cereus versus B. anthracis) was not even a consideration. Inlight of immunoassays, it is reasonable to consider the use of a bindingagent, such as an antibody, to selectively bind a target analyte, suchas an antigen to achieve species, subspecies, strain or serotypeselectivity. The concept was first demonstrated by coating a roughenedsilver electrode with anti-human thyroid stimulating hormone antibody(anti-TSH) to capture the TSH antigen (Tarcha et al U.S. Pat. No.5,266,498; 1993). However, “no” SERS of this binding event was shown.Antibody capture was only demonstrated when a second anti-TSH antibodyfunctionalized with a dye (2-[4′-hydoxyphenylazo]-benzoic acid) wasadded, which itself attached to the bound TSH antibody to produce aspectrum of the dye. Furthermore, the laser wavelength was matched tothe absorption of the dye to generate resonance Raman scattering, which,like SERS, is known to enhance Raman scattering by as much as six ordersof magnitude. Yet this combined surface-enhanced Raman and resonanceRaman spectroscopic (SER(R)S) approach only achieved a modestly lowconcentration detection of 4 microg/mL (4×10⁻⁶ International Units/mL)for the TSH antigen when 40 microg/mL (10 times the concentration) ofthe dye-anti-TSH complex was added. This result is questionable, sincethe addition of 40 microg/mL of the dye-anti-TSH complex to a samplecontaining “no” TSH antigen produced a more intense dye spectrum,suggesting that the spectrum was due more to the dye being in directcontact with the metal surface as opposed through binding to the TSHantigen in forming a dye-(anti-TSH)-TSH-(anti-TSH)-silver complex. Inany case, the authors stated that no spectra of antibodies or antigenswere obtained, but only spectra of a dye, and antigen-antibody bindingtypically required an hour or more (two hours for the above example).

In the 19 years since the Tarcha patent there have been only a fewpublications in which a biochemical of interest, such as those describedabove, has been successfully bound to an antibody attached to a SERSsubstrate and detected by SERS. This is due to the fact that the targetanalyte must be within the plasmon field of the surface-enhanced Ramanactive (SER-active) metal to achieve a significant enhancement of theRaman signal, since the enhancement decreases with distance to the12^(th) power. This means that the plasmon field extends 10 nm at most.The challenge, and lack of sensitivity, has been elegantly demonstratedfor the binding of E. coli to an antibody coated on silver nanoparticles(Naja et al., “Raman-based detection of bacteria using silvernanoparticles conjugated with antibodies”, Analyst, 132, 679 (2007). Itwas found that the signal was enhanced only by a factor of 20, far lessthan the expected 1 million or so, even when a very small antibody,Protein A, brought the bacteria to a distance of 8 nm from the metalsurface. Furthermore, the E. coli sample was allowed to bind (incubate)to the antibody functionalized silver particles “overnight”, and driedon a glass slide to concentrate the sample prior to SERS measurements.Clearly, the basic concept of using SERS as the detector forimmunoassays does not provide sufficient sensitivity or speed for theapplications discussed above.

To overcome the sensitivity limitation, a number of researchers havefollowed the approach of Tarcha et al, by adding dye molecules to theimmunoassay. White et al. (U.S. Pat. No. 6,750,065) describe the use ofantibodies that bind drug-dye complexes, such that the introduction ofthe drug will displace the drug-dye complex, which can be detected“downstream” again using a combined surface-enhanced Raman and resonanceRaman spectroscopy approach. The patent details methods to synthesizethree drug-dye complexes, but provides no information regarding theantibody nor data showing displacement or the drug-dye complex or thepatented principle. Sun et al. (U.S. Pat. No. 7,485,471) describe theuse of a dye-labeled antibody that binds a specific antigen, which inturn binds a second antibody that contains a seed particle that, inturn, is used to grow a SER-active particle, which will ideally interactwith the dye. No data are supplied, such as the time required to growthe SER-active particle or how the dye will come to interact with it.Consequently, neither of these patents addresses the rapid decrease insignals due to distance from the metal nor the long binding times. Infact the value of using a dye label to increase sensitivity has beenquestioned by Zhang et al. (“Protein adsorption drastically reducessurface-enhanced Raman signal of dye molecules”, J Raman Spectrosc, 41,952, 2010), who have shown that the SER signal intensity of dye-proteincomplexes added to silver colloids is “reduced” by several orders ofmagnitude compared to the dye by itself. They showed that a flouresceindye attached to either bovine serum albumin, lysozyme, trypsin, orconcanavalin A produced “no” SER signal. Furthermore, they suggest thatthe signals observed for the other dyes used in their experiments mayhave resulted from the dyes coming into direct contact with the silvercolloids, as the proteins were not bound to the silver colloids beforeintroduction of the dye.

Based on the foregoing, it is believed that one of ordinary skill in theart would not expect a significant SER signal to be generated from anantibody-antigen pair bound to SER-active particles (especially when theantigens are micron-sized bacterial cells), even with the introductionof additional SERS particles after binding has been achieved; nor wouldthe signal strength be expected to be sufficient to detect as few as 10bacterial spores or CFUs; nor would it be expected that detection couldoccur in less than 20 minutes, i.e. without a long incubation time.

SUMMARY OF THE INVENTION

It is the broad object of the present invention to provide a novelmethod and apparatus for detecting, identifying, analyzing, andquantifying target analyte(s), i.e., chemical, biochemical, orbiological substances, in test samples.

It is a more specific object of the invention to provide such a methodand apparatus wherein detection and analysis are effected bysurface-enhanced Raman spectroscopy, with substantial selectivity,sensitivity, and speed, through the use of target analyte-specificbinding agents and multiplicative signal enhancement.

It has now been found that the foregoing and related objects of theinvention are attained by the provision of a method and apparatus inwhich an analyte-specific binding agent is attached to at least one of afirst SER-active material, of which a support structure is comprised,and a second SER-active material comprising a liquid reagent. An analytesample is added to one (or both) of the functionalized SER-activematerials, and the liquid reagent is added to the support structure suchthat the SER-active materials are effectively attached to the targetanalyte so as to act in concert to cooperatively produce greatlyenhanced SER signals.

More specifically, certain objects of the invention are attained by theprovision of a method for the detection, identification, analysis andquantitation, by surface-enhanced Raman spectroscopy, of at least onedesignated target analyte, comprising the steps: providing a supportstructure comprised of a first, SER-active metal-containing SER-activematerial; providing a liquid reagent comprised of a second, SER-activemetal-containing SER-active material; functionalizing the SER-activemetal of at least one of the first and second SER-active materials withat least one binding agent that has a specific capability for bindingthereto at least one designated target analyte, to provide at least onefunctionalized SER-active material; obtaining an analyte samplesuspected of containing the at least one designated target analyte;adding the analyte sample to one of the at least one functionalizedSER-active materials; establishing or maintaining conditions sufficientto effect attachment of the at least one designated target analyte tothe at least one binding agent of the one functionalized SER-activematerial; effecting the removal of any unbound chemicals, biochemicals,or biologicals from the one functionalized SER-active material to whichthe at least one target analyte is attached; adding the liquid reagentto the support structure; establishing or maintaining conditionseffective to cause the other of the first and second SER-activematerials to attach to the at least one target analyte already attachedto the at least one binding agent of the one functionalized SER-activematerial; irradiating the first and the second SER-active materialsattached to the at least one target analyte so as to cause theSER-active metals of the SER-active materials to cooperatively generatea SER spectrum; detecting the cooperatively generated SER spectrum; andanalyzing the detected SER spectrum to determine the presence andquantity of the at least one designated target analyte.

In preferred embodiments the effects of the SER-active metals of thefirst and second SER-active materials, in cooperatively generating theSER spectrum, are synergistic. That is, multiplicative enhancement, thatis quite superior to the sum of the enhancements of the two individualSER-active materials (both of which have been shown to produceinsignificant enhancement [vide supra and infra]), are unexpectedlyproduced.

One or both of the SER-active materials can be used in solution to carryout the method of the invention. However, in most cases one SER-activematerial will be attached to a support structure to which the otherSER-active material is added. Furthermore, in most cases the supportedSER-active material will normally be functionalized with the bindingagent, but alternatively the added SER-active material could befunctionalized and, in some cases, both SER-active materials could befunctionalized.

The support structure employed in the method will usually comprise asubstrate, supporting the “first” SER-active material, that isfunctionalized with the “at least one” binding agent. The substrate maybe fabricated from metal, glass, paper, plastic, or any suitablematerial that may be in the form of a substantially planar sheet orplate, or membrane, or (in the case of glass and plastic) in the form ofa wall of a vial, capillary, channel, or the like. A substantiallyplanar sheet or plate may, in some instances, desirably be formed with amultiplicity of wells for receiving the analyte sample or samples. Theplanar sheet or plate may, in some instances, be porous so as to form amembrane capable of passing the sample through it.

The “first” SER-active material attached to a substrate will generallybe of a form selected from: metal colloids as isolated spheres,clusters, aggregates, monolayers, multilayers, ring or tube structuresdeposited on the substrate; metal-coated particles, monospheres, orlithographically produced structures, such as posts or pyramidalstructures on the substrate; metal depositions of islands on thesubstrate; electrochemically generated metal surfaces; structuredefining holes in metal surfaces; metal grown structures on thesubstrate, including porous metal structures; and metal-doped porousmaterials. Specific examples include gold and silver colloids, silvercoated on a monolayer of polystyrene monospheres, gold-coatedlithographically produced silica posts, evaporated silver islands onglass, copper electrodes, silver-doped sol-gels, nickel-coatedpolystyrene beads on support structures, and gold-doped swellablepolymers.

Preferably, the first SER-active material will be composed of at leastone SER-active metal and at least one porous material. The lattermaterial will be sufficiently porous to readily pass the “at least one”binding agent, the “second” SER-active material, the “at least one”designated target analyte, and any signature chemical that may bepresent. In certain instances, the porous structure will advantageouslybe effective to separate or extract the “at least one” designated targetanalyte from other components of the analyte sample.

In some preferred cases the binding agent will be attached to the metalof a SER-active material using a linker chemical, chemicalfunctionality, or biochemical; specific examples include cysteine, athiol group, or N-hydroxy succinimide esters. In other preferred casesthe binding agent will be attached to the metal of a SER-active materialusing a spacer chemical to improve performance of the binding agent byspacing the binding agent a sufficient distance above, or otherwise awayfrom, the SER-active metal surface to bind the target analyte withoutinterference or restriction from the metal surface. For example, it maybe beneficial to use a spacer chemical to extend a peptide, as thebinding agent, above the surface by several angstroms, such that thebinding agent has room to bind the target analyte. Exemplary spacerchemicals are aliphatic thiols, which can self-assemble on theSER-active material to form a monolayer. In those instances the lengthof the aliphatic chains determines the distance between the SER-activemetal surface and the binding agent, which will typically be between 2and 5 angstroms. Another strategy to increase the distance between theSER-active metal and the binding agent involves adding terminal groupson the binding agent (e.g., amino acid residues for proteins andpeptides, and bases for oligonucleotides).

In certain embodiments it may be useful to add a blocking agent to aSER-active surface. Such an agent will serve to block the adsorption ofpotentially interfering chemicals, biochemicals, or biologicals,preferably without itself producing a SER spectrum. Examples of suitableblocking agents are ethanolamine, polyethylene glycol, polylysine, andbovine serum albumin.

Each of the “first” and “second” SER-active materials employed in themethod of the invention will usually comprise a metal selected from thegroup consisting of copper, gold, silver, nickel, platinum, rhodium,iron, ruthenium, cobalt, nickel, palladium, and alloys and mixturesthereof. The metal of the “first” SER-active material will normally beof particulate form or in the form of a surface having a morphology thatis functionally equivalent to metal particles so as to generate aplasmon field when irradiated. The metal of the “second” SER-activematerial will normally be of particulate form, and will advantageouslyhave an electropotential that is effective to attract it to the bound“at least one” target analyte. It will be appreciated that the “first”and “second” SER-active materials may (or may not) be different from oneanother, and that the “first” SER-active material may comprise a mixtureof at least one SER-active metal and at least one porous material.

The “at least one” designated target analyte and the “at least one”binding agent will normally be a chemical, biochemical or biologicalsubstance, and will usually be paired with one another for effectiveinteractions; such pairs may include (a) antibodies and antigens, (b)peptides and biologicals, (c) drug receptors and drugs, (d) enzymes andtheir specific biochemical substrates (e.g. inhibitors and cofactors),(e) carbohydrates and lectins, and (f) nucleic acid sequences and theircomplements.

In some embodiments of the present method the “at least one” targetanalyte will be a bioagent, food or waterborne pathogen, or adisease-causing pathogen. As the term is used herein, “bioagents”include, but are not limited to, Bacillus anthracis (including Ames andUT500 strains), Brucella melitensis subspecies and serotypes (e.g.abortus, suis), Clostridium botulinum A, Ebola virus, Francisellatularensis, Leishmania genus, Marburg virus, Mycobacterium leprae(leprosy), Puumala hantavirus, Ricin toxin, Variola virus (small pox),and Yersinia pestis.

Food and waterborne pathogens referred to herein, include, but are notlimited, to Aeromonas hydrophilia, Bacillus cereus, Campylobacterjejuni, Clostridium botulinum B and C, Clostridium difficile andperfringens, Cryptosporidium parvum, Escherichia coli (O157:H7), Giardialamblia, Hepatitis A, Listeria monocytogenes and subspecies andserotypes, Salmonella enterica and subspecies and serotypes, includingtyphimurium, Shigella dysenteriae and subspecies and serotypes, Vibriocholera, and Yersinia enterocolitica,

Disease-causing pathogens referred to herein include, but are notlimited to, Burkholderia mallei (glanders), coronaviruses (Severe AcuteRespiratory Syndrome (SARS)), Corynebacterium diphtheriae, Dengue fever,Enteric viruses, Enterobacter aerogenes, Equine encephalitis,hemorrhagic fevers, Hepatitus (A-E), herpes simplex viruses 1 and 2,human immunodeficiency virus, Legionella, Lyme borreliosis (disease),measles, meningitis, mumps, MRSA, Mycobacterium tuberculosis, Mycoplasmapneumonia, Neisseria gonorrhoeae, Norwalk virus, Ortho-myxoviridaefamily (influenza), Plasmodium genus (Malaria), rabies virus,rhinoviruses, Rickettsia species (e.g. rickettsii, prowazekii, typhi),Rotovirus, Rubella virus, saxitoxin, sepsis, Shigella, and subspeciesand serotypes, dysenterial, Staphylococcal (enterotoxin B, agalactiae,and pyogenes), Streptococcus pneumonia, Swine disease, Treponemapallidum (syphilis), Varicella zoster virus (chicken pox, shingles),West Nile virus, and Yellow fever.

In certain embodiments the method will include a further step of addingto the analyte sample at least one reagent that is effective, under theconditions existing or established, for releasing a signature chemical,a signature biochemical, or a signature biological, with the signaturechemical, biochemical or biological thus constituting the “at least one”designated target analyte. More specifically, the “at least one” targetanalyte may be a released signature chemical selected from the groupconsisting of calcium dipicolinate, dipicolinic acid, mono-protonateddipicolinic acid, deprotonated dipicolinic acid, diaminopimelic acid,n-acetyl-muramic acid, ribonucleic acid, phosphoglyceric acid, andsulfoacetic acid. Examples of released signature biochemicals includeamino and nucleic acids, nucleotides, nucleosides, deoxyribonucleicacid, ribonucleic acid, gene sequences, plasmids, phages, peptides,proteins, lipids, polysaccharides, haptans, antibodies, antigens,biomarkers, enzymes, steroids, hormones, lectins, aptamers, includingfragments or polymers thereof (e.g. antibody-fragment, polypeptides).

The reagent for effecting release of a signature chemical, biochemical,or biological, will normally be an acid, base, or solvent, or mixturethereof. Example acids include acetic, adipic, ascorbic, citric, formic,fumaric, lactic, malic, palmitic, peracetic, propionic, salicylic,sorbic, succinic, and trihaloacetic acids, while example solventsinclude acetone, acetonitrile, benzene, chloroform, carbontetrachloride, cyclohexane, dichloromethane, diethyl ether,dimethylsulfoxide, ethyl acetate, ethylene glycol, isopropyl ether,methyl ethyl ketone, n-hexane, phenol and its derivatives,tetrahydrofuran, toluene, and mixtures thereof.

Additional objects of the invention are attained by the provision ofapparatus, in the form of a kit of components, for use in the detection,identification, analysis, and quantitation of at least one designatedtarget analyte in an analyte sample, by surface-enhanced Ramanspectroscopy. The apparatus comprises: packaging means for thecontainment of a multiplicity of components (i.e., normally containingall components of the kit); at least one SER-active sample devicecomponent constructed for receiving the at least one designated targetanalyte, and for operative assembly with a Raman spectrometer to enabledetection, identification, analysis, and quantitation, bysurface-enhanced Raman spectroscopy, of at least one designated targetanalyte; at least one collection component that is constructed foranalyte sample collection and discharge; at least one containercontaining a liquid reagent comprised of the second SER-active material;additional containers containing reagents; a component for introducingthe liquid reagent into the SER-active device component; and optionallyadditional components, such as sample transfer components, mixingcontainers and filtering apparatus, as needed. The apparatus willusually comprise a plurality of collection components contained by thepackaging means, such as eyedroppers, syringes, pipettes andmicropipetters, and swabs, or a multiplicity of such componentsassembled in various combinations. Preferably, the kit will additionallyinclude a water-supply component comprised of a container containingdistilled, deionized water, at least one mixing container, and afiltering component for filtering an analyte sample. In most instancesthe apparatus will additionally include at least one second reagentsupply component, comprised of a container containing, for example, abuffered solution to maintain a pH, or an agent to degrade a biologicalmaterial to effect release of a signature species, such as a signaturechemical, a signature biochemical, or a signature biological; in thelatter case, the signature species will normally constitute the “atleast one designated target analyte,” as more specifically identifiedherein.

The SER-active device component of the instant apparatus is constructedto enable irradiation and collection of Raman scattered radiation by andfrom a Raman spectrometer. It provides a support structure comprised ofthe first SER-active material, the SER-active metal of which may befunctionalized with at least one binding agent which has the specificcapability of binding thereto the at least one designated targetanalyte, and that is accessible for the deposit thereupon of analytesamples, the second SER-active material, and any additional reagentsemployed; it may advantageously comprise a vial, capillary, disc,multi-well plate or lab-on-a-chip (LOC) device. The disc or multi-wellplate may be made of a porous membrane, so as to pass the sample throughit. As previously noted, rather than functionalizing the metal on asupport structure, or in addition thereto, the metal of the SER-activematerial comprising the liquid reagent may be functionalized with atleast one binding agent.

As also indicated above, a linker or a spacer chemical or biochemicalwill, in some instances, preferably be interposed between the bindingagent and the associated metal of a SER-active material for attachingthe at least one binding agent thereto, and thus may be a feature of theapparatus of the invention; the first and second surface-enhanced Ramanactive materials employed therein will comprise the metals hereinaboveand hereinafter specified. The surface-enhanced Raman active materialcomprising the support structure may be of particulate form, or in theform of a surface having a morphology functionally equivalent to metalparticles, so as to generate a plasmon field when irradiated; the metalof the second surface-enhanced Raman active material will preferably beprovided as a colloidal suspension of metal particles in the form ofisolated spheres, clusters, aggregates, ring or tube structures. Inaddition to providing SER activity, the metal particles (or equivalentstructure) will desirably also function by reason of having anelectropotential that is effective to attract it to the bound designatedtarget analyte. As indicated above, the metals of the first and secondsurface-enhanced Raman active materials may be different from oneanother.

As used in the present application, the following terms and referencesare to be understood to have the meanings hereinafter set forth, unlessa different or further definition is provided, or the context makes itclear that another meaning is intended:

“Chemical substance” means any general chemical, including drugs,explosives, radionuclides, pesticides, inorganic or organic pollutants,and their associated precursors or break-down products (e.g. hydrolysisproducts, metabolites, etc.).

“Biochemical substance” means any biochemical involved in chemicalprocesses of living organisms, including amino and nucleic acids,nucleotides, nucleosides, peptides, proteins, lipids, polysaccharides,haptans, antibodies, antigens, biomarkers, enzymes, steroids, hormones,lectins, aptamers, phages, prions, immunoglobulins, toxins, includingfragments or polymers thereof (e.g. antibody-fragment, polypeptides).Specific examples include serum albumin, immunoglobulin G, human thyroidstimulating hormone, and prostate specific antigen.

“Biological substance” means a microbial life form, such as any algae,bacteria, fungi, protozoa, or virus.

As used in respect of a target analyte, “identify” means to determinethe chemical, biochemical, or biological identity of the target analytefrom cooperatively generated spectra.

“Specificity,” as used in respect of a target analyte, refers to theselective binding of a particular target analyte, as opposed to analyteswith similar structure that would produce a false positive response.Examples include: (1) selective binding of B. anthracis, but not otherbacteria, especially not clostridia and most especially not otherbacilli; (2) E. coli O157:H7, but not other bacteria, and mostespecially not other E. coli; and (3) or selective binding of cocaine,but not other drugs, and especially not cocaine metabolites, such asbenzoecognine.

Exemplary of the sensitivity that is achieved using the method andapparatus of the invention is the ability to detect (i.e., to obtain SERscattering data) and measure 2500/m³ B. anthracis spores in air, 100CFUs/mL E. coli in lettuce, 10⁶ CFUs MRSA/mL sputum, and 10 ng/mL of adrug in saliva.

The test sample (i.e., a sample containing a target analyte) utilized incarrying out the method of the invention may be obtained from a broadvariety of gaseous, liquid, and solid sources. Gas sample sourcesinclude, but are not limited to, air, a gaseous chemical, a chemical orbiological aerosol, exhaust fumes, ventilation system or room air,exhaled or ventilated breath, extracted lung air, and mixtures thereof;examples of target analytes in specific gas test samples includebiological spores or toxic industrial chemicals in air, and bacteria ordisease biomarkers in exhaled breath.

Liquid sample sources include, but are not limited to, chemicals, water,and biological fluids such as blood plasma, blood serum, whole blood,exhaled breath condensate, nasal mucus, saliva, semen, spinal fluid,sweat, throat sputum, and urine. Examples of target analytes in liquidtest samples include a drug in a chemical solvent; bacteria orpesticides in a drink, such as juice or milk; bacteria in a lake, sewer,or water-treatment sample; a pollutant in a lake, river, ocean, groundwater, or rain sample; a drug in blood plasma, saliva, or sweat; abacterium in nasal mucus, saliva, or throat sputum; and a diseasebiomarker in semen or urine.

Examples of solid sample sources include, but are not limited to, food,soil, an animal part, feces, a frozen material, and a substance on asurface. Examples of target analytes in solid test samples includebacteria and pesticides in or on fruits, meats, and vegetables;pesticides in soil; poisons in animal kidneys and livers; bacteria infeces; bacterial spores on a mail-sorting machine; explosive materialson or in an improvised explosive device; and drugs and explosives onclothing, luggage, hair, fingertips or fingernails.

The volume of the test sample employed will be defined by the requiredanalysis, and can be quite large or very small. For example, cubicmeters of air would normally be collected for the purpose of detectingaerosolized bacterial spores, whereas a drop of saliva would normallysuffice for the detection of a drug. The volume of the analyte sampleemployed will generally be quite small, such as 10 mL; often, however,the volume will not exceed 1 mL, and in many instances it will be muchless.

For many test samples the method will desirably include the further stepof treating a collected or sampled material or substance (e.g., a testsample) so as to separate the “at least one” target analyte from othercomponents, and to thereby produce an “analyte sample.” The residual“other” components will normally constitute all chemicals, biochemicals,and biologicals present in the collected or sampled material that mayinterfere significantly with analysis. Such interferences includehindering flow of the target analyte(s) to the binding agents,deactivating the SER-active materials, and/or producing spectra thatwould substantially prevent the spectrum of the at least one targetanalyte from being observed. Such a further treating step may includethe use of an extracting or degrading chemical effective to make the “atleast one” target analyte available, and means for separating the atleast one target analyte from the chemical used for extracting ordegrading.

In the case of solid test samples, the extracting or degrading chemicalmay be selected from the group consisting of acids, bases, solvents, andcombinations thereof. Examples include acetic acid to releasedipicolinic acid (DPA), a signature for endospores, and a chloroform andphenol mixture to release DNA from bacteria, DPA and DNA being asignature chemical and a signature biochemical for these pathogens,respectively.

In the case of body fluids, the extracting or degrading chemical may beselected from the group consisting of solvents, acids, bases, mucolyticagents, surfactants, and mixtures thereof. Suitable mucolytic agentsinclude N-acetyl-L-cysteine (NALC), Amboxol, Bromhexine, andcombinations thereof; a solution of NALC and NaOH is presently preferredfor separating drugs from saliva.

In some cases a combination of reagents may be used. For example,mucolytic agents used to separate B. anthracis spores from sputumfollowed by the use of acetic acid to release dipicolinic acid.

In the case of many gaseous, chemical, and water test samples, thetarget analyte will inherently be available for separation, and anextracting chemical need not be used. Specific examples includebacterial spores in air, drugs in a solvent, and pesticides in water.

Target analytes, and other components, may be mutually separated fromthe produced degrading chemical solution using a chemical, physical, orchromatographic method.

Chemical treatment of a sample may employ a solvent for the at least onetarget analyte, which solvent will desirably be of such polarity as torender it capable of extracting the target analyte. Suitable solventsinclude water containing appropriate acids and bases for pH adjustment;organic liquids such as acetone, acetonitrile, benzene, chloroform,carbon tetrachloride, cyclohexane, dichloromethane, diethyl ether,dimethylsulfoxide, ethyl acetate, ethylene glycol, isopropyl ether,methyl ethyl ketone, n-hexane, phenol and its derivatives,tetrahydrofuran, and toluene; and mixtures of such solvents.

Physical treatment for effecting mutual separation may involve passageof the sample through a filter. Suitable filters comprise poroussubstrates such as paper, coated paper, paper fibers, polymer, polymerfibers, mixed paper and polymer fibers, cellulose acetate, glass wool,cotton, diatomite, porous glass, sintered glass, zirconia-stabilizedsilica, derivatized silica-based matrices, sol-gels, and derivatizedsol-gels. They may also comprise a supported membrane covered withseparation materials, such as the silica gels, zirconia-stabilizedsilica, derivatized silica-based matrices, sol-gels, derivatizedsol-gels, glass beads, long-chain alkane particles, derivatizedlong-chain alkane particles, polymers, derivatized polymers,functionalized membranes, alumina, polystyrene, dendrimers, immobilizedcrown ethers, and ion-exchange resins.

Chromatographic methods may employ the same separation materials, andwill desirably utilize a liquid carrier solvent for at least one of thetarget analytes.

The “first” SER-active material referred to herein will desirably beattached to a support structure that provides a solid surface, asdescribed. It will, in general, consist of a SER-active metal in theform of particulates or coatings on surface or in matrices or solutions,or other appropriately sized structures that can generate a surfaceplasmon field when irradiated. Effective forms of SER-active metals canbe produced by chemical or electrochemical etching, by photolithographicprocess, by vapor or chemical deposition, by reduction of metal salts,or by other means known to those skilled in the SERS arts.

The added “second” SER-active materials will generally be of a formselected from metal colloids or monospheres, or metal coated spheres insolution as isolated spheres, clusters, aggregates, or ring or tubestructures. Specific examples include silver colloids, aggregates ofcolloids, gold-coated polystyrene spheres, and gold-coated magnetic ironbeads.

The SER-active metal of at least one of the “first” and “second”SER-active material should, in any event, be readily functionalized witha suitable binding agent; i.e., a chemical, biochemical, or biologicalsubstance that binds, or attaches, to a specific target analyte. Suchfunctionalization generally includes the formation of a chemical bond orphysical interaction, including covalent, ionic, hydrogen bonds, or vander Waals or electrostatic forces, between the metal and the bindingagent.

Porous materials that contain SER-active metals and that comprise the“first” SER-active material can be produced by chemical synthesis, usingmethods that include sol-gel chemical syntheses employing silica-basedalkoxides and SER-active metals, and polymer syntheses employinghydrophilic monomers that allow the inclusion of SER-active metals. Sucha SER-active material may also be a porous volume produced by mixing aporous medium and a SER-active metal; such media include sol-gels,silica gels, silica stabilized by zirconia, derivatized silica-basedmatrices (e.g., trifunctional quanternary amine, aromatic sulfonicacid), long-chain alkane particles (e.g., C8 to C18), derivatizedlong-chain alkane particles (e.g., phenyl, cyano, etc.), and otherporous media commonly known to one skilled in the art of porous media.Preferably, such a metal-doped porous material will be sufficientlyporous to permit liquids (e.g., the target analyte, and liquid reagentscomprising the “second” SER-active material) to be transportedtherethrough to the first SER-active metal sites. Such a porous materialmay advantageously be of a chemical composition that is effective toextract a target analyte from a test sample (treated as necessary), tothereby improve sensitivity (i.e., the effective detection of a targetanalyte by SER scattering, in the concentration present).

Desirably, an alkoxide is used to produce a polar-positive,polar-negative, or non-polar sol-gel that is effective in extracting theappropriately charged or neutral target analyte. Suitable alkoxidesinclude but are not limited to tetramethyl orthosilicate, tetraethylorthosilicate, methyl-trimethoxysilane, methyltriethoxysilane,aminopropyltriethoxysilane, aminopropyltrimethoxy-silane,octadecylsilane, etc. Additional selectivity for extraction can beimparted using polymers (e.g. polydimethylsiloxane for extractions usinghydrophobic interactions, and polyethylene glycol (PEG) for extractionsusing hydrophilic interactions).

A non-functionalized SER-active material, used in the practice of thepresent invention, will desirably have an electropotential that iseffective to attract it to the target analyte that is attached to thefunctionalized SER-active metal. Examples of materials having such anelectropotential include electropositive silver colloids, effective tobe attracted to negatively charged target analytes, target analytefunctional groups, or target analyte surface sites; and electronegativegold coated polystyrene spheres, effective to be attracted to positivelycharged target analytes, target analyte functional groups, or targetanalyte surface sites.

Binding attachment may occur through chemical bonds or physicalinteractions, such as covalent, ionic, or hydrogen bonds, or by van derWaals or electrostatic forces between charged, polar, hydrophobic, orhydrophilic chemical groups on the surface of the at least one analyteand binding agent. Binding usually occurs within a period of 60 minutesafter introduction of an analyte sample to a functionalized SER-activematerial, under conditions sufficient to effect the necessaryinteraction; preferably, however, attachment will occur within a periodof 10 minutes, and most preferably within a 5-minute period. Thesebinding times are indicative of the speed with which the steps of thepresent method can be effected.

As previously indicated, the method of the invention will, usuallyinclude a wash step, using a suitable solvent, for effecting removal ofany unbound or unwanted chemicals, biochemicals, or biologicals that mayinterfere significantly with the measurement that is to be made. Suchsuitable solvents include water having a selected pH value (e.g. using abuffer), acetone, acetonitrile, benzene, chloroform, carbontetrachloride, cyclohexane, dichloromethane, diethyl ether,dimethylsulfoxide, ethyl acetate, ethylene glycol, isopropyl ether,methyl ethyl ketone, n-hexane, tetrahydrofuran, toluene, and mixturesthereof. The wash step will usually require a period of no more than 30minutes, preferably no more than 5 minutes, and most preferably no morethan 1 minute.

The second SER-active material is added to the first promptly after thedesignated target analyte has been allowed to attach to thefunctionalized SER-active material (whether it is on a support structureor in a liquid reagent). When a wash step is employed, addition of thesecond SER-active material will most desirably be performed as soon asit is feasible to do so; usually within 10 minutes, and preferablywithin 1 minute, and most preferably as soon as the wash step iscomplete. These time periods are also indicative of the speed with whichthe instant method can be carried out.

The support structure will desirably be so constructed as to effectivelyenable irradiation of the target analyte, combined with the first andsecond SER-active materials, and the collection of the cooperativelygenerated spectrum. Irradiation is preferably effected using a laser,and the generated SER signal is detected by a Raman spectrometer. TheRaman spectrometer may be scanning or optical filter, preferablyinterferometric or dispersive in design, and capable of producingspectra using a strip chart recorder, a plotter, or preferably acomputer with appropriate software.

The objective of analyzing the detected spectra, to determine at leastthe presence of the target analyte in the sample, will normally beachieved based on the spectral peak positions of the target analyte,when a SER spectrum matches, automatically or manually, a previouslymeasured spectrum of the target analyte, which most often will be storedin a spectral library. In some instances, the spectral differenceswithin a genus between two species, subspecies, strains or serotypes,will be subtle and chemometrics will be used to identify the particularspecies, subspecies, strains or serotypes; augmentation of the method bythe application of chemometric techniques can also assist in determiningpathogenicity, potency, and toxicity and viability. Chemometrics,suitable for the present method, will typically involve measuringnumerous spectra of the species, subspecies, strains or serotypes to bedifferentiated and applying statistical analysis to the spectra so thatdifferences can be modeled, then using the model to categorize anunknown measured target analyte as a particular species, subspecies,strain or serotype.

Quantitation of the target analyte (i.e., determining the amountpresent) is achieved by measuring the SER spectral intensity. Spectralintensity can be a peak height or area, or a combination of peak heightsor areas, in some cases compared by ratio to a spectral intensity of aninternal or external reference material. Such an internal reference willpreferably be a solvent in which a target analyte is contained, or thesignal produced by the binding agent. An external reference may be achemical added specifically for the purpose of providing a reference, orsoftware that compares the spectral intensity to a previously measuredconcentration sample set.

Needless to say, sample collection and treatment, addition of the sampleto the SER-active sample device, effecting a wash step, addition of thesecond SER-active material, addition of any release reagent, irradiatingthe structure, detecting the SER scattering, and analyzing the acquiredspectrum to detect, identify, analyze, and quantify the target analyte,will all desirably be performed with substantial selectivity,sensitivity, and speed. Those desiderata are enabled by the presentinvention, as described herein.

With further regard to the kit of components embodying the apparatus ofthe invention, such components will usually include, in general terms,means for obtaining a test sample; means for treating the sample toeffect mutual separation of at least one target analyte and interferingchemicals, biochemicals, or biologicals, to produce an analyte sample;means for introducing the analyte sample to a support structure thatcontains a first SER-active material; means for introducing a washsolution to remove any unbound chemicals, biochemicals or biologicals;means for adding a second SER-active material; and, optionally, meansfor adding an additional reagent to make available signature chemicals,biochemicals or biologicals. Apparatus embodying the invention may alsoinclude means for irradiating the support structure and target analyteto generate a spectrum; a spectrometer for collecting and detecting thegenerated spectrum; and means for analyzing the spectrum to detect,identify, analyze, and quantify the target analyte, if present.

The support structure comprising the apparatus may advantageouslyinclude at least one section for combining the analyte sample with abinding agent, the wash solution, the second SER-active material and, ifappropriate, a signature chemical releasing agent. In certainembodiments, such a section for combining will be in direct liquid flowcommunication with the means for sample treating; it may either bephysically separated from the section by which radiation is enabled, orthe two functions may be performed by a single component. In someembodiments it may be desirable to detect and quantify more than onetarget analyte. In such cases the support structure employed may containmultiple sections, each having a first SER-active material, which may befunctionalized with analyte-specific binding agents; multiple sectionsmay take the form of wells in a multi-well plate; multiple channels in alab-on-a-chip (LOC), either in sequence or in parallel; or multiplesegments, in sequence, within a capillary.

The optical interface between the SER-active sample device and the Ramanspectrometer will of course be designed to enable irradiation of theSER-active metals and target analyte, and collection of the generatedRaman scattering in a manner appropriate to the device. Examples includea simple lens, a cylindrical lens, a microscope objective, a fiber opticprobe, etc. A single lens can be used to focus the laser to a point inthe device and to collect the Raman radiation. This would be appropriatefor a SER-active sample device having a single measurement point, suchas a SER-active disc, capillary, vial, or lab-on-a-chip. A single lenssystem could also be used to measure multiple points on a SER-activesample device, such as a capillary, lab-on-a-chip or multi-well plate,by either moving the lens or the SER-active device, manually orautomatically, to align the lens with each measurement point. In someinstances a cylindrical lens may be appropriate to allow measurement ofmultiple points, and hence multiple target analytes, simultaneously on aSER-active sample device. For example, the cylindrical lens could beused to focus the excitation laser in the shape of a line across severalparallel channels of a LOC, or wells of a multi-well plate, each channelor well being designed to detect a unique target analyte, and to collectthe SER scattered radiation, such that each measurement point isspacially separated along one axis of a two dimensional array detector,the other axis being used to obtain the Raman spectrum as a function ofwavenumbers (i.e. wavelength).

Ideally, the support structure and analyzer afford ease of use andportability, for at-site measurements, such as at the site of a bioagentattack or at the bedside of a hospitalized patient.

Exemplary of the utility of the present invention are the following,generalized examples:

General Example A

A surface, representative of surfaces associated with mail facilities(bins used to hold letters, sorting equipment, etc.), hospitals, and thelike, is examined to detect potential contamination by bioagents orpathogens. A drop of solvent (i.e., about 50 microliters), such aswater, is applied to the surface so that the bioagents or pathogens canbe collected in solution and delivered, after being passed through afilter, if appropriate, to a first SER-active material, functionalizedwith a binding agent, attached to a SER-active sample device. Afterallowing time for the bioagents or pathogens, if present, to bind to thebinding agent (typically, a period of five minutes), a wash solution isdrawn through or across the SER-active sample device to remove anyunbound chemicals, biochemicals, or biologicals that may be present.Then a second SER-active material, in solution, is drawn into or ontothe SER-active sample device. In the case of Bacilli or Clostridiaspores, the addition of the second SER-active material may include, orbe followed by, a weak acid solution, such as acetic acid, to cause therelease of calcium dipicolinate into the solution, and thereby to formdipicolinic acid. The SER-active sample device is then placed within, orattached to, the sample compartment of any suitable Raman spectrometer(as described herein), and the required measurement is performed.

Since the amount of solvent used in the present procedures wouldgenerally be known, that information can be used, in conjunction withknowledge of the measured surface area covered, to quantify, in terms ofsurface area (e.g. 10 spores per cm²), the bioagents or pathogensdetected. As an alternative, a swab could be used to wipe a predefinedarea, the swab then being introduced into a predefined volume of solventso as to again to enable quantification of the measurement.

A variety of devices can be employed to dispense the solvent, to collectthe resultant sample solution, and to deliver the solution to theSER-active sample device. For example, a simple eyedropper, a disposablepipette, a calibrated micropipetter, or a calibrated syringe could beused both to dispense the solvent and also to collect the samplesolution; alternatively, a different device, such as a syringe fittedwith a particle and/or chemical filter, could be used for collection.

General Example B

In a second manner of use of the present method, air samples aresemi-continuously or periodically collected and examined for bioagentsor pathogens, and any apparatus that is commonly used to collect andconcentrate particles from the air can serve as a sample-acquisitiondevice. In many instances, however, cyclone or impactor devices will bepreferred, which may be designed to handle large volumes of air, at highflow rates, and to collect particles within certain size ranges, such asbiological particles of 1-20 microns. In any event, such an airbornesample acquisition device would advantageously deposit the collectedparticles into a vessel containing a solvent, to allow treatment andintroduction to a SER-active sample device, as previously described. Itwill also be appreciated by those skilled in the art that wet-walledcyclones, designed to efficiently capture particles and transfer them toan underlying container, could be employed to particularly goodadvantage; the solvent used for sample collection could then serve asthe wall-washing solution.

To enable continuous monitoring, collected bioagents or pathogens couldbe deposited upon a rotating carousel holding numerous containers ofsolvent, into a solvent stream, or upon a moving tape to which thesolvent is added. In the first case, the content of each container couldbe analyzed as described above, manually or automatically. In the caseof a solvent stream, the sample could be passed through a filter toremove interfering or extraneous collected particles, and then passedinto a series of capillaries or channels within a LOC in a series ofsteps; e.g., stopping the flow to allow binding; washing the surface;introducing the second SER-active material; making SERS measurements,and then resuming the flow, generally to an unused LOC. In the case of amoving tape, an appropriately placed source of solvent could be added toremove the bioagents or pathogens from the tape, and deliver theresultant sample to the support structures described above, with theappropriate steps, such a, a wash step, a step of introduction of asecond SER-active material, a release agent, etc. In any event, theSER-active sample device, such as capillaries, LOCs, or multi-wellplates, would be aligned, or otherwise associated, with the irradiatingsource of any suitable Raman spectrometer, to enable the requiredmeasurement to be performed.

General Example C

In another mode of use of the instant method, the targetanalyte-containing sample is obtained from blood, blood plasma, feces,nasal mucus, saliva, semen, sweat, throat sputum, urine, or otherbiological matter; examples of typical target analytes are hospitalpathogens, disease biomarkers, drugs, and drug metabolites. Samples areacquired using appropriate means, e.g., a swab for collecting sweat, andadditional means may be desirably employed to separate the pathogensfrom the body fluid prior to introduction of the sample to the supportstructure. When, for example, a swab is used to collect a sample, anon-cotton swab (e.g., having a polyester absorbent element) willgenerally be preferred because cotton fibers tend to plug pores ofseparation media. The swab, or other implement used for collection, isweighed before and after collection to determine the sample mass, andthereby allow results to be quantified.

Nasal mucus, saliva, and throat sputum are composed largely of water(about 95%) and mucins, otherwise known as glycoproteins (about 0.5-2%)(Kaliner, M, Z Marom, C Patow, “Human respiratory mucus”, J Allergy ClinImmunol, 73, 318-323, 1984). The mucins consist of a protein core (about20%) with oligosaccharide side chains (about 80%), crosslinked bydisulfide and hydrogen bonds, which give mucus, saliva, and sputum theirhigh viscosity.

To best enable analysis of nasal mucus, saliva, and throat sputum,mucins should be broken down, or “liquefied,” to separate the targetanalyte, such as spores or drugs, that may be present (see for example;Kubica, G, A Kaufmann, W Dye, “Comments on the use of the new mucolyticagent, N-acetyl-L-cysteine, as a sputum digestant for the isolation ofmycobacteria”, Am Rev Respir Dis, 87, 775-779, 1964; or Murray, P, EBaron, J Jorgensen, M Pfaller, R Yolken, Manual of ClinicalMicrobiology, 5^(th) ed, ASM Press, Wash. D.C., 1995). Nasal mucus,saliva, and throat sputum may be chemically degraded, to allow mutualseparation of the target analyte(s) and mucin degradants, usingmucolytic agents, surfactants, acids and/or bases. Suitable mucolyticagents include N-acetyl-L-cysteine (NALC), Amboxol, Bromhexine, or anycombination thereof; in many instances, a solution of NALC and NaOH willpreferably be employed. The surfactant utilized may advantageously belithium dodecyl sulfate, sodium dodecyl sulfate, or a combinationthereof, and an acid or base employed may be HCl, H₂SO₄, HNO₃, NaOH, orKOH, or any combination consisting of such acids or bases.

Nasal mucus, saliva, and throat sputum degradants and the target analytemay be separated by chemical, physical, or chromatographic methods, orany combination thereof. Chemical methods will typically employ asolvent to extract the target analyte from the degradants, such as waterhaving a selected pH value, acetone, acetonitrile, benzene, chloroform,carbon tetrachloride, cyclohexane, dichloromethane, diethyl ether,dimethylsulfoxide, ethanol, ethyl acetate, ethylene glycol, isopropylether, methanol, methyl ethyl ketone, n-hexane, tetrahydrofuran,toluene, and mixtures thereof; mixtures that aid in selectivelyextracting the target analyte into a desired solvent are especiallydesirable.

Physical separation methods will typically employ a filter that capturesthe degradants while allowing the passage of the target analyte.Suitable filters comprise a porous substrate or other element, such aspaper, coated paper, paper fibers, polymer, polymer fibers, mixed paperand polymer fibers, cellulose acetate, glass wool, cotton, diatomite,porous glass, sintered glass, zirconia-stabilized silica, derivatizedsilica-based matrices, sol-gels, and derivatized sol-gels.

Chromatographic techniques suitable for use will typically employ aseparation material, contained in a column, through which the degradantsand target analyte pass at different flow rates. Such chromatographicseparation materials include silica gels, zirconia-stabilized silica,derivatized silica-based matrices, sol-gels, derivatized sol-gels, glassbeads, long-chain alkane particles, derivatized long-chain alkaneparticles, polymers, functionalized membranes, alumina, polystyrene,dendrimers, immobilized crown ethers, and ion-exchange resins.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic illustration showing apparatus embodying thepresent invention, depicted in a series of stages comprising a methodembodying the invention.

FIG. 2A is a diagrammatic illustration of an analytical kit embodyingthe present invention;

FIG. 2B is a diagrammatic, very much simplified illustration, takengenerally along lines B-B of FIG. 2A and drawn to a greatly enlargedscale, of a substrate carrying a SER-active material depositfunctionalized with a binding agent having a specific capability forbinding a target analyte.

FIG. 3 is a diagrammatic illustration showing components of apparatusembodying the invention, depicted in a series of steps comprising amethod embodying the invention, for measuring a pathogen sample on asurface by SERS.

FIG. 4 is a diagrammatic illustration showing components of apparatusembodying the invention, depicted in a series of steps comprising amethod embodying the invention, for measuring a food sample containingfoodborne pathogens by SERS.

FIG. 5 is a diagrammatic illustration showing components of apparatusembodying the invention, depicted in a series of steps comprising amethod embodying the invention, for measuring infectious diseasepathogens by SERS.

FIG. 6 is a series of four SER spectra obtained for 1000 B. cereusspores in a 10 μL sample introduced into four different 1 mm glasscapillaries prepared as follows: A) containing B. cereus specificpeptides functionalized on silver in a sol-gel matrix with silvercolloids added after the sample and a wash step; B) identical to A), butno colloids were added; C) identical to A), but no silver particlesfunctionalized with peptide were incorporated in the sol-gel; and D)identical to C), but acetic acid was included in the silver colloidsolution. The intensities of the B) and C) spectra have been multipliedby 10, while the intensity of the D) spectrum has been multiplied by100.

FIG. 7 comprises two SER spectra obtained for two different samples, A)containing 100 B. cereus and 10,000 B. subtilis spores in a 10 μLsample, and B) containing only 10,000 B. subtilis spores in a 10 μLsample, both introduced into identical 1 mm glass capillaries containingB. cereus specific peptides functionalized on silver in a sol-gelmatrix. Both samples were allowed to bind for 5 minutes, washed withwater, and further treated by addition of a silver colloid solutioncontaining acetic acid to generate spectrum A and spectrum B,respectively.

FIG. 8 comprises two SER spectra obtained for 5000 E. coli O:157H:7 CFUsin a 50 μL sample added to two 1-inch glass disc coated with E. coliO:157H:7 specific antibodies functionalized on gold colloids. In bothcases the samples were allowed to bind for 10 minutes, washed withwater, air dried, and then, in the case of spectrum A but not in thecase of spectrum B, further treated by the addition of a silver colloidsolution.

FIG. 9 comprises two SER spectra of 10⁷ CFUs of A) L. monocytogenes andB) L. innocua on generalized Listeria antibody functionalized gold dopedin a sol-gel matrix.

FIG. 10 is a plot of Principle Component Scores for 30 Listeria samples,consisting of 15 known L. monocytogenes (open circles) and 15 known L.innocua (open squares) and two correctly identified unknown samples(solid circle and solid square).

DETAILED DESCRIPTION OF THE PREFERRED AND ILLUSTRATED EMBODIMENTS

As indicated above, the present invention provides a novel method andapparatus to detect, identify, analyze, and quantify at least one targetanalyte, in a test sample that binds to target analyte-specific bindingagent attached to a first SER-active material. A second SER-activematerial that binds to the binding agent is added, and the twoSER-active materials act together, and in concert, to providemultiplicative signal enhancement for detection and analysis by SERS,affording substantial selectivity, sensitivity, and speed.

Turning now in detail to FIG. 1, therein illustrated, in each of thefour depictions (A through D), is a substrate 1, such as may be made ofmetal, glass, paper, plastic or other suitable material, and may be awall (i.e., an inside surface) of a vial, capillary or channel, or aplate or other structure described herein, coated with the firstSER-active material 2 that is, in turn, functionalized with a bindingagent 3. The binding agent 3 is attached to the SER-active material 2 bylinking or spacing molecules 4. A blocking agent 5, attached to anyunfunctionalized portion of the metal of the first SER-active material,is used to prevent any extraneous SER-active chemicals, biochemicals, orbiologicals contained in the sample from interacting with the metal toproduce an interfering SER spectrum.

In use, the analyte sample containing the target analyte 6, which mayhave been obtained by pretreatment of a test sample, is deposited on, ordrawn across or through, the functionalized structure (1, 2, 3, 4, 5) byappropriate means, and allowed time to bind to the binding agent (shownas a second stage, in depiction B); preferably, the binding time is onthe order of minutes. Any unbound or unwanted chemicals, biochemicals,or biologicals are then removed from the surface using a wash solution(not illustrated, but typically a component of the apparatus depicted inFIG. 2A), and a second SER-active material 7 is then introduced upon thesurface (depiction C) so as to attach to the target analyte 6 (depictionD). The thus prepared and treated substrate is placed in an appropriatesample holder of a Raman spectrometer (not shown in this figure), bywhich the surface-enhanced Raman spectrum of the sample is recorded.Computer driven software then analyzes the spectrum to determine if thetarget analyte(s) are present and, if so, in what quantities.

FIG. 2A illustrates a kit that includes essential components of theapparatus of the present invention, contained in a case 9 of suitableconstruction. The contents of each kit will of course correspond to theapplication for which it is intended, but a main component is aSER-active sample device having an appropriate substrate coated with afunctionalized gold- or silver-doped sol-gel, or other SER-activematerial, as described in the present specification. Such SER-activesample devices include internally coated or filled glass capillaries 10,a lab-on-a-chip device 12 having three internally coated or filledchannels 14, a coated disc 16, or a coated multi-well plate 18.

As seen in FIG. 1 and FIG. 2.B, in all instances the SER-active devicecomponent includes a substrate 1, coated with a SER-active material 2,functionalized with a binding agent 3. It will be appreciated that thesubstrate 1 is generalized and will, as present in each of the severalrepresentative SER-active sample devices shown in FIG. 2A, have suchcurvature or other shape, and length, width, radial, etc. dimensions, asmay be presented by the surface upon which the SER-active material andbinding agent are to be attached.

In addition, the kit will contain the appropriate reagents contained inbottles or other containers, as necessary to carry out the method of thepresent method. However, a second primary component is a SER-activeliquid reagent, such as a gold, silver, or other suitable metal colloid,as isolated spheres, clusters, aggregates, ring or tube structures, incontainer 20. Other reagents that may be necessary or desirable includewater, in container 22, used in collecting samples and to wash awayunbound chemicals in the substrates after the binding step; bufferedsolutions to collect nasal mucus or the like; digesting agents, such asNALC, to liquefy certain samples; and acetic acid, or the like, todegrade biologicals, as necessary. It will be appreciated thatcontainers for the latter solutions, agents, and reagents are notillustrated, but will be of course be of suitable size and constructionto best accommodate their contents. It will also be appreciated that thecontainers will advantageously have associated means for introducingtheir contents into the SER-active sample device included in the kit,such means being integral with the container (e.g., a normally closedspout), or separate therefrom but adapted for use therewith (e.g.,syringe 23, a pipette 24, or the like).

A primary function of syringes 23, pipettes 24, and like components ofcourse is to transfer liquids; they may for example be used to dispensewater or other liquid onto a surface, to collect the possible pathogenspresent, or to introduce a sample into a SER-active sample device.Pipettes 24, syringes 23, and filter holders 32 included in the kit willdesirably be constructed to connect to the corresponding includedSER-active sample device. Connectors may however be included; forexample, syringe ports may be provided for attachment to LOCs.Plunger-mounted swab 26 may be used to collect saliva or throat sputum,and its associated barrel 28 may be used to discharge the sample into asample container, such as a vial 30. A filter holder 32, and replaceablefilters 34, may be employed in either collecting or transferring asample, as required, and additional vials will be supplied, asnecessary, for sample collection, transfer, or mixing; even a blender(not shown) may be included in the kit for blending a sample, such as offood, with an extracting reagent, as may be necessary.

FIG. 3 shows the steps for measuring a pathogen sample on a surface bySERS, using a capillary that has been internally coated or filled withmetal-doped sol-gel functionalized with an aptamer, antibody, peptide,protein, or the like. In accordance with step A, a reagent 35, such aswater contained in a syringe 23, is squirted upon a surface S containingsuspected pathogens P (i.e., the target analyte). In step B the reagentcontaining the target analyte (pathogens P), comprising the sample, isdrawn back into the syringe. The sample is then injected, in Step C,into the capillary 10 containing the functionalized, metal-dopedsol-gel, and allowed to bind for 5 minutes. A second syringe 23 a isthen used, in step D, to pass water through the capillary 10, therebyremoving any unbound material. Then in step E a third syringe 23 b isused to introduce metal colloids 7 into the capillary 10, in a volumesufficient to coat or fill the sol-gel portion. It is noted that it maybe advantageous to add a reagent, such as acetic acid, to cause a markerchemical, such as dipicolinic acid, to be released before, with, orafter the addition of the colloid 7. In step F the capillary 10 ismounted above the measurement point of a Raman spectrometer 36.

FIG. 4 shows the steps for measuring a food sample containing foodbornepathogens, by SERS, using a metal, glass, paper, plastic or othersuitable material to form a planar substrate, such as the disc shown,coated with an antibody-functionalized SER-active material. In step A, afood sample F, potentially containing foodborne pathogens P as thetarget analyte, is placed in a blender 38 containing appropriatereagents 37. After blending, the sample is extracted into a syringe 23,in step B, and passed through a filter set 32, 34 to remove anyundesirable chemicals and to deposit pathogens P onto a disc 16 coatedwith the functionalized SER-active material. In this case the sample isallowed to bind to the antibody for 20 minutes. A second syringe 23 a,containing an appropriate reagent 39, is then used to gently wash thedisc to remove any unbound material. Next the sample is dried, in stepD, by blowing air onto it using an air hose 40. A third syringe 23 b isthen used, in step E, to introduce the second SER-active material 7,which could be silver colloid aggregates or ring structures, orgold-coated polystyrene or iron monospheres, onto the disc 16 in avolume sufficient to coat or fill the SER-active material deposit.Finally, in step F, the disc 16 is mounted below the measurement pointof a Raman microscope system 36.

FIG. 5 shows the steps for measuring infectious disease pathogens bySERS using a lab-on-a-chip device having channels coated or filled withfunctionalized SER-active material, as described herein. As a first stepA, a patient sample H, such as of saliva, nasal mucous, or throat sputum(containing suspected disease pathogens P), is collected using, forexample, a swab 26, and is added, through a cooperating barrel 28, tovial 30 that contains a reagent 41 for liquefying the mucans of whichsuch samples are comprised. After 2 minutes of mixing, thepathogen-containing reagent is drawn, in step B, by syringe 23 through afilter set 32, 34 through which the degraded mucans and undesirablechemicals are passed, but which captures the pathogens. In step C, asecond syringe 23 a, containing an appropriate reagent 43, is used tocarry the captured pathogens into a lab-on-a-chip device 12. The LOC hasthree channels (in the illustrated embodiment), each functionalized withdifferent antibodies or peptides designed to capture differentpathogens; for example, channels 44, 46, 48 could be built to bind MRSA,TB, and NP, respectively. The channels may be arranged in parallel, asshown, or in series; i.e., comprised of a single channel with threedifferent functionalized SER-active material segments. A third syringe23 b, containing an appropriate reagent 45, is then used in step D towash the channels 44, 46, 48 so as to remove any unbound material; afourth syringe 23 c, is used in step E to introduce the secondSER-active material, such as silver or gold colloids 7 in a volumesufficient to coat or fill the SER-active material containing portionsof the channels; and a fifth syringe, 23 d, may be used to control flowof the sample and reagents into the channels 44, 46, 48. Finally, instep F, the lab-on-a-chip device 12 is mounted below the measurementpoint of a Raman spectrometer 36 having the capability of scanning thethree channels (or segments, as the case may be). It should be notedthat the LOC employed herein can contain mixing chambers, controllablevalves, and reagent wells, as described in U.S. Pat. No. 7,713,914,particularly noting FIG. 14 therein.

Exemplary of the efficacy of the present invention are the followingspecific examples:

Example One B. Cereus Detected in a Peptide Functionalized Silver-DopedSol-Gel Coated Capillary

The silver-doped SER-active sol-gels employed in the filled glasscapillaries utilized in this example were prepared in accordance withthe method of Farquharson et al. (U.S. Pat. No. 6,623,977), whichcapillaries were filled with silver-doped sol-gels in accordance withthe techniques described by Farquharson et al. (“Rapid dipicolinic acidextraction from Bacillus spores detected by surface-enhanced Ramanspectroscopy” Applied Spectroscopy, 4, 351-354, 2004). In essence, asilver amine complex (Ag(NH₃)₂ ⁺OH⁻), consisting of a 5:1 v/v solutionof 1 N AgNO₃ and NH₄OH (28 wt % NH₃ in H₂O), was mixed with an alkoxideconsisting of a 2:1 v/v solution of methanol and tetramethylorthosilicate in a 1:8 v/v silver amine:alkoxide ratio. A 10 microLaliquot of the forgoing mixture was then drawn into a 1-mm diameterglass capillary to fill a 10-mm length. After sol-gel formation, theincorporated silver ions were reduced with dilute sodium borohydride,followed by a water wash to remove residual reducing agent.

Next, the silver particles contained in the sol-gel were functionalizedwith a peptide binding agent designed to specifically bind B. cereus.Functionalization was accomplished by attaching a cysteine residue tothe C-terminus of the peptide and adding it to the silver-doped sol-gel.Cysteine serves as a link, as it forms a covalent bond to the silverbetween its thiol group and silver. It also serves as a spacer, in thatthe peptide is displaced from the silver surface by about 2-10angstroms, providing space therebetween within which the spores caneffectively bind. The functionalized sol-gel was then washed with asolvent to remove any unbound peptide.

A first set of experiments, designated Part 6A through Part 6D, wasperformed to illustrate the concerted, multiplicative effect of usingtwo SER-active materials in accordance with the present invention; thedata obtained are graphically presented as Spectra A through D in FIG.6:

Part 6A

A 10 microL sample containing 1000 B. cereus spores was injected into acapillary, as described above, using a syringe. The volume of the samplematched the volume defined by the diameter of the capillary and thelength coated with peptide-functionalized silver-doped sol-gel. A periodof 5 minutes was allowed for the sample to bind (it is noted that insome instances it is advantageous to draw more of the sample through thecapillary, to introduce more spores to the binding sites, but at a slowrate to allow binding). A 20 microL wash solution was drawn through thecapillary to remove any unbound or unwanted chemicals, biochemicals, orbiologicals. Then a solution containing silver colloids, prepared inaccordance with Lee and Meisel (“Adsorption and Surface-Enhanced Ramanof Dyes on Silver and Gold Sols”, J. Phys Chem., 86, 3391-3395, 1982),was injected by syringe into the capillary, again matching the volumedefined by the capillary diameter and SER-active coating length. Thecapillary was placed in a capillary holder of an FT-Raman spectrometer,and the SER spectrum of the sample was recorded in 1 minute using 75 mWof 785 nm laser power. The entire measurement, from sample introductionto spectral analysis, took less than 10 minutes. The resultant spectrum,designated A, in FIG. 6, shows the unique spectrum of dipicolinic acidwith major peaks at 815, 1008, 1382 and 1428 cm⁻¹, which identify thepresence of the B. cereus spores. It is worth noting that thesignal-to-noise ratio (S/N) for the 1008 cm⁻¹ peak is ca. 600,suggesting a detection limit based on a S/N of 3 equivalent to 5 B.cereus spores.

Part 6B

Another 10 microL sample containing 1000 B. cereus spores was injected,by syringe, into the capillary of a second, identical silver-dopedSER-active sol-gel filled glass capillary, prepared identically to theone described in Part 6A above. The same procedure was used, but nocolloids were added to the capillary after the sample wash. Theresultant SER spectrum, designated B in FIG. 6, shows no indication thatthe spores are present, and hence the use of a first SER-active materialfunctionalized to bind B. cereus alone was insufficient to produce a SERsignal.

Part 6C

In a third part of this experiment an identically prepared capillary wasagain used, but with the silver amine complex being omitted. The sol-geldid not therefore contain any silver particles, nor could it befunctionalized with the peptide. Nevertheless, a sample and silvercolloids were added at the same concentrations as in the Part 6Ameasurement, but with the wash step being omitted to avoid removal ofthe spores and colloids. The resultant SER spectrum, designated C inFIG. 6, shows no indication that the spores are present, whichdemonstrates that the addition of a second SER-active material, heresilver colloids, was insufficient to produce a SER signal.

Part 6D

To establish that the colloids and spores were in fact present in thesample of Part C, the same experiment was repeated once again, but withacetic acid added to the analyte sample. The resultant spectrum,designated D in FIG. 6, contains the characteristic DPA peaks,confirming the presence of the spores and colloids, but at an intensityabout 100 times less than that which was produced in the initialmeasurement, spectrum 6A.

Part 7A

A second set of experiments was performed to illustrate the selectivityof the peptide binding agent; the data obtained are graphicallypresented as Spectra A and B in FIG. 7:

A 10 microL sample containing 100 B. cereus and 10,000 of B. subtilisspores was injected into a capillary, again prepared as describedpreviously in this example; the volume of the sample matched the volumedefined by the diameter of the capillary and the length coated withpeptide-functionalized silver-doped sol-gel. A period of 5 minutes wasallowed for the sample to bind, following which a 20 microL washsolution was drawn through the capillary to remove any unbound orunwanted chemicals, biochemicals, or biologicals; in this case, the B.subtilis spores are unwanted biologicals. Then a solution containingsilver colloids was injected by syringe into the capillary, once againmatching the volume defined by the capillary diameter and SER-activecoating length, but in this case the silver colloid solution alsocontained acetic acid to promote the rapid release of dipicolinic acid,as discussed above and disclosed in U.S. Pat. No. 7,713,914. Thecapillary was then placed in a capillary holder of an FT-Ramanspectrometer, and the SER spectrum of the sample was again recorded in 1minute using 75 mW of 785 nm laser power. The resultant spectrum,designated A in FIG. 7, shows the unique spectrum of dipicolinic acidwhich, assuming the binding was specific, identifies the presence of theB. cereus spores, as is verified by the next part of this experiment.

Part 7B

A second 10 microL sample containing only the 10,000 B. subtilis sporeswas measured using a procedure identical to Part 7A. The resultantspectrum, designated B in FIG. 7, is seen to contain only a weak aceticacid peak at 880 cm⁻¹ and two weak peptide peaks at 660 and 1180 cm⁻¹,with dipicolinic acid peaks no longer being present. This, and themeasurement of the previous part, demonstrates that the peptide does notbind B. subtilis but selectivity binds B. cereus.

Based on the relative concentrations for the 100-spore and 1000-spore B.cereus samples measured using the two SER-active materials acting inconcert, versus use of the colloids alone, and the correspondingintensities of the DPA peak at 1008 cm⁻¹ shown in spectrum A of FIG. 7and spectrum D of FIG. 6 (the FIG. 7A 1008 cm⁻¹ spectral peak intensityequals 100 times the 6D spectral peak), the additional signalenhancement achieved by the method and apparatus of the presentinvention is about 1000 times. It will be appreciated that this resultis completely unexpected, based upon the knowledge of those of ordinaryskill in the art and upon previously published measurements described inthe literature.

As will also be appreciated, in the case of an unknown sample, thenumber of B. cereus spores can be quantified by comparing the intensityof any of the primary peaks, such as the ring breathing mode at 1008cm⁻¹ to the intensity of the Cys-Ag stretching peptide mode at 660 cm⁻¹,so long as a relationship between the peak intensity ratios to sporecount has been established. The acetic acid solvent peak, at 880 cm⁻¹,can be used to the same purpose of quantifying the number of sporespresent.

It should of course be realized that the invention, as illustrated bythe present example, would be of particular benefit to homeland andmilitary security activities when used to selectively detect B.anthracis, such as by the detection of spores in ambient air, in mail,on surfaces, or even in nasal mucus, throat sputum, or saliva.Furthermore, it will be appreciated that the method and apparatus of theinvention could be expanded to include all bioagents, provided that anappropriate binding agent, such as a peptide, an aptamer, or anantibody, was employed.

Example Two Measurement of E. Coli Detected on an AntibodyFunctionalized Gold Colloid Coated Glass Slide Part 8A

A gold colloid solution was prepared according to Pal et al.(“Photochemically prepared gold nanoparticles: A substrate for surfaceenhanced Raman scattering,” Current Sci., 84, 1342-1346, 2003). One-inchdiameter glass discs were spin-coated at 2500 rpm by adding 1 mL of thegold colloid solution drop-wise to form a monolayer. Once the coatingdried, a layer of multi-functional polyethylene glycol ligands—a linker,containing two thiol groups for anchoring onto the gold and a terminalN-hydroxysuccinimide ester group for coupling to antibodies, was addedto the surface. After 4 hours the discs were washed with water to removeany unbound PEG from the gold surface. The E. coli selective antibodywas then added to the surface and allowed to functionalize over a periodof 2 hours, and another buffer wash was thereafter used to remove anyunfunctionalized antibody. Then a solution of polylysine was added tothe disc to block any of the gold surface not functionalized with theantibody, and the resulting structure was also washed with a buffer toremove any polylysine that might attach to the antibody. Previousmeasurements of adenine, with and without the blocking agent, produced aSER spectrum of adenine only when the blocking agent was not used, thusverifying the effectiveness of the blocking agent.

Part 8B

A sample of lettuce was inoculated with 1,000,000 E. coli colony-formingunits. The test sample was blended in water to produce an approximately100,000 E. coli CFU per 1 mL lettuce-water solution. A 1 mL sample waspushed through a filter to remove the degraded lettuce while passing theCFUs, to produce an analyte sample, 50 microL of which was depositedonto the antibody-functionalized gold colloid-coated glass slide. Afterallowing a 20-minute binding period, the surface was gently washed witha reagent to remove any undesired or unwanted chemicals, biochemicals,or biologicals. The sample was then force dried with blowing air,followed by the addition of 50 microL of a silver colloid solution,during a period of about 3 minutes. The glass slide was placed at thefocal point of a Raman microscope and recorded in 1 minute using 75 mwof 785 nm laser excitation. The resultant spectrum, designated A in FIG.8, shows two peaks at 740 or 1340 cm⁻¹ indicative of E. coli. Onceagain, the same sample was measured identically, but without theaddition of colloid. The resultant spectrum, designated B in FIG. 8,only shows the SERS of the antibody, a doublet at 640 and 665 cm⁻¹,again demonstrating the importance of the second SER-active materialacting in concert with the first. Furthermore, the intensity ratios ofthe E. coli peaks in spectrum A to the antibody peaks in spectrum B, canbe used to determine that the 50 microL sample contained ca. 5000 E.coli CFU, provided that a correspondence between the ratio of knownconcentrations has been established.

It should be noted that in some cases it is important to differentiatepathogen species, subspecies, strain or serotype, but unique peptides orantibodies for each species, subspecies, strain or serotype are notalways known, only generic binding agents are known. It may still bepossible however to identify the species, subspecies, strain or serotypeif the Raman spectra are different, but the differences in the spectramay be subtle, requiring the use of chemometrics, statistics applied tochemistry.

As an example, fifteen 5000 CFUs per 50 microL samples each of Listeriamonocytogenes and Listeria innocua were prepared and measured on fifteenseparate SER-active sample devices as described for E. coli, butfunctionalized with a generic Listeria antibody as the binding agent.FIG. 9 shows the SERS of the averaged spectra for the two species withinthe Listeria genus; modest spectral differences are apparent. Softwarewas used to develop a simple discriminate linear regression model usingprincipal component analysis that employed a series of weighted spectralregions that correlate to the two different Listeria species, as shownin FIG. 10.

Then two additional discs were prepared as above, functionalized withthe generic Listeria antibody, and used to measure two samplesdesignated unknowns in FIG. 10, one containing L. monocytogenes and onecontaining L. innocua. The spectra were then analyzed in terms of thePrincipal Component Scores, which placed them within the species regionsallowing the identification of the Listeria species contained in eachsample.

It should be realized that such chemometric-based relationships can bedeveloped for a number of target analyte properties as part of theanalysis of the present invention, such as pathogenicity, potency,toxicity, and viability. Other linear regression models, such asprincipal component regression analysis, partial least squares,classical least squares, inverse least squares, or even higher-ordermodels might of course also be used to develop such a relationship.

Example Three Measurement of Hospital Pathogens: Methicillin ResistantStaphylococcus Aureas (MRSA), Tubercles Bacillus (TB), and NosocomialPneumonia (NP)

As described hereinabove in connection with FIG. 5, a lab-on-a-chipdevice is designed with three channels, each containing a SER-activemetal functionalized with a different binding agent (peptide, aptamer,antibody, etc.) for selectively binding MRSA, TB or NP. In someinstances the SER-active metal is chemically attached to the walls ofthe channels; in other cases it is contained in the channel, preferablyincorporated in a sol-gel. A throat sputum sample, collected from ahospital patient, is treated with reagents, such as N-acetyl-L-cysteineand sodium hydroxide, to liquefy the mucans that potentially contain thetarget hospital pathogens, as described in U.S. Pat. No. 7,713,914. Thesample is filtered to trap the pathogens, while passing the mucans andother potentially interfering biochemicals, after which the pathogensare collected and added to a solvent. The resultant pathogen sample isthen injected or drawn into the LOC, by syringe or other means, in suchmanner that it is equally divided among the three channels and so that,if present, the pathogens bind to their respective binding agents. Awash solution is injected or drawn through the three channels of the LOCto remove any unbound pathogens or potentially interfering chemicals orbiochemicals, following which a solution containing silver colloids isinjected or drawn through the channels so as to optimize, acting inconcert with the resident SER-active metal within the channels, the SERsignal ultimately generated, in accordance with the present invention.Thus, the LOC is placed on a sample stage constructed so as to becapable of aligning each channel with the Raman excitation laser, andthe SER spectra are recorded. Software is used to analyze the spectraand determine if any of the three pathogens are present and, if so, inwhat quantity.

It will be readily apparent to those skilled in the art that the methodand apparatus described herein can be utilized to detect and quantifyother pathogens contained in body fluids, such as cholera, hepatitis,human immunodeficiency virus, influenza, malaria, and strep throat, aswell as alternatives to the numerous other target analytes specificallymentioned herein. For example, the method and apparatus can be used toidentify biomarkers in body fluids, indicative of a disease ordeleterious health state, such as for the detection of antigen 3 inurine, indicative of prostate cancer, or cholesterol in blood,indicative of hardening of the arteries.

As noted previously, the three channels of the LOC described could bereplaced with a single channel containing each of the binding agents,functionalized on metal particles, spaced in sequence; or the bindingagents could all be in a single sol-gel mixture. In the former case, butnot the latter, a positioning stage would still be required; in thelatter case, however, spectral analysis could be used to deconvolute thecontributions from each bound pathogen, so long as the spectrum for eachpathogen is unique. Needless to say, an LOC employed herein can have anysuitable number of channels, and/or sequential deposits, and/or bindingagent species, as dictated by the objectives of the analysis andpractical considerations. In certain embodiments, moreover, an LOC cancontain liquefying reagents, filters, and SER-active colloids inreservoirs, as indicated, for example, in FIG. 14 of U.S. Pat. No.7,713,914.

Example Four Measurement of Usher Syndrome (Blindness and Deafness) Genein Parental DNA

Prior to having children, many parents, especially those with a familyhistory of health problems, have DNA testing performed to determine therisks of passing on a disease. The present example addresses thatconcern.

A monolayer of polystyrene latex spheres of uniform 40 nm diameter sizeare spin coated onto a glass disc, dried, and overcoated with a thinlayer of silver using a thermal evaporation chamber, as described by VoDinh, et al. (Surface-enhanced Raman spectrometry for trace organicanalysis”, Anal. Chem., 56, 1667, 1984). The silver is thenfunctionalized with oligonucleotide #1 via an aliphatic thiol thatserves to both link the oligonucleotide to the metal and as a 2-10angstrom spacer to provide sufficient room above the metal surface sobinding can occur unhindered. The oligonucleotide can consist of anynumber of nucleotides bound together, but preferably they are relativelyshort, containing 10 to 20 units; such nucleotides are often referred toas DNA or RNA aptamers. A solution of bovine serum albumin is added tothe functionalized silver substrate, which binds to any exposed silver,blocking the surface from extraneous SER-active chemicals, biochemicals,or biologicals contained in the analyte sample that might interact withthe metal and produce an interfering SER spectra.

SER-active gold colloids that are also magnetic are prepared accordingto the procedure of Mosier-Boss (“Surface-enhanced Raman spectroscopysubstrate composed of chemically modified gold colloid particlesimmobilized on magnetic microparticles”, Anal. Chem., 77, 1031, 2005).In essence, gold colloids are bound to amine-terminated iron oxidemicroparticles and, after several hours of mixing, a magnet is used toseparate the gold colloid-coated iron oxide microparticles.Oligonucleotide #2 is attached to those particles via an aliphatic thiolthat, as above, serves to both link the oligonucleotide to the metal andprovide unhindered binding room.

Both aptamers are compliments to two different DNA targets containedwithin the MY07A gene, which is associated with Usher Syndrome. Apurified DNA sample, obtained from prospective parents, that has beenfragmented using restrictive enzymes or other techniques known to thoseskilled in the art, is added to the aptamer-functionalized silversubstrate (as a SER-active sample device), and the MY07A gene fragments,if present, are allowed to bind i.e. hybridize, for 15 minutes at 37° C.The silver substrate is washed to remove any unbound sample. Thefunctionalized gold micro-particles are then added to the functionalizedsubstrate. A magnet, placed below the silver substrate, is used to speedthe interaction between the oligonucleotide #2 and its complement on theMY07A gene fragments bound to the substrate via oligonucleotide #1, ifpresent. The magnet is removed, and the substrate is washed to removeany unbound gold particles. The SER-active sample device is placed inthe sample compartment of a Raman spectrometer, and the spectrum ismeasured and analyzed.

If the MY07A gene is not present, then either a relatively weak spectrumof oligonucleotide #1 is obtained, or if a complement to oligonucleotide#1 is present, but not due to the MY07A gene, then a weak spectrum ofthe complimentary, hybridized sequence is obtained. If however the MY07Agene is present, then an intense spectrum of both complimentedoligonucleotide #1 and #2 is obtained. The dramatic increases insensitivity, attributable to the use of a second SER-active particle inaccordance with the present invention, and in selectivity due to the twosequences, allows performing this measurement in as brief a period as 20minutes, substantially faster than the current 2 to 6 hours' timerequired by PCR.

Again, the present invention could be applied to the detection of allgenetic diseases for which the genetic sequences are known or becomeknown. Furthermore, as described above, an LOC could be designed todetect any of numerous genetic diseases.

Example Five Drug Discovery Using a Multi-Well Microplate

Drug discovery involves methods that screen the activity of numerouspotential drugs by examining their interaction with a disease or healthtarget which, in accordance with the present invention, would be thebinding agent. In such cases the binding agent could be a simplenucleotide or a protein which represents the binding site for potentialdrugs designed to cure a disease. For example, the binding agent couldbe an enzyme involved in cancer growth which, if inhibited by the drug,would arrest the cancer.

In accordance with this Example, each well of a 384-well microplate isbottom-coated with a first SER-active material by introducing thereinto,and drying, a 10 microL gold colloid solution. A solution containing abinding agent then is added to each well. A linking species, such as asulfur containing chemical or biochemical, may be added to the bindingagent so that it forms a covalent bond with the gold surface. One of asmany as 384 potential drugs, in a solvent such as water, is added toeach well, followed by flushing, using 10 microL solvent, to remove anyunbound drugs. Then a 10 microL silver colloid solution is added to eachwell. The microplate, as the SER-active sample device, is thereafterplaced on a sample stage that is capable of aligning each well with aRaman excitation laser, and the SER spectra are recorded. If no drug ispresent only a weak binding agent SER signal will be observed. If,however, a drug binds to a protein binding site, then an intense SERsignal of the potential drug or it's metabolites will be produced evenat exceptionally low concentrations, such as 1 ng/mL.

Here again it will be appreciated by those skilled in the art that themethod and apparatus described in this Example could be used todiscover, in addition to drugs, many important chemicals, biochemicalsor biologicals. For example, a microplate filled with a particularpathogen as the binding agent could be used to examine a series ofpeptides or antibodies so as to identify a peptide or antibody thatbinds to the pathogen.

Example Six Identification of Effective Antibiotic and Antiviral Drugsfor Pathogens

The approach described in Example Five is readily adapted for theidentification of effective antibiotic and antiviral drugs forpathogens. To do so, for example, each well of a 96-well SER-activemicroplate is functionalized with a binding agent designed to capture atarget pathogen (e.g. methicillin resistant Staphylococcus aureus, H1N1virus, Plasmodium falciparum, etc.). To each such functionalized well,10 microL of a pathogen suspension is added and allowed to attach to thewell surface for 15 minutes, after which the wells are flushed with abuffer solution to remove any unbound pathogen. Then 10 microL aliquots,of as many as 96 potential drugs in their respective solvents, areadded, one each to a well, and the drug solutions are allowed to bind tothe target pathogen for 30 minutes, for example. The wells are thenwashed with a solvent to remove any unbound drug molecules, followingwhich a 10 microL silver colloid solution is added to each well. Themicroplate is placed on a sample stage and, as previously described, SERspectra are recorded, but with each well being measured for 5 seconds,in sequence, thus requiring a total of only 8 minutes' time to measurethe 96 wells. This procedure is repeated through a period of 2 hours,for example, thereby providing 15 time-dependent spectra for each wellso as to efficiently monitor the effectiveness of each drug.

For drugs that are effective against the target pathogen, not only willthey produce intense SER spectra of the drugs or their metabolites,indicating binding, but changes in the pathogen spectral fingerprintsare observed over time. If the drug molecules are not effective, thepathogen spectral features would not change over time.

Thus, it can be seen that the present invention provides a novel methodand apparatus for detecting, identifying, analyzing, and quantifying, ina test sample, target analyte(s) that bind to target analyte-specificbinding agents. More specifically, it provides such a method andapparatus wherein analyses are effected by surface-enhanced Ramanspectroscopy, with substantial selectivity, sensitivity, and speed,through multiplicative signal enhancement.

1.-31. (canceled)
 32. Apparatus, in the form of a kit of components, foruse in the detection, identification, analysis, and quantitation of atleast one designated target analyte in an analyte sample, bysurface-enhanced Raman spectroscopy, comprising: packaging means for thecontainment of a multiplicity of components; a SER-active devicecomponent constructed for receiving the at least one designated targetanalyte, and for operative assembly with a Raman spectrometer to enabledetection, identification, analysis, and quantitation, by SERS, of theat least one designated target analyte, said SER-active device providinga support structure comprised of a first, SER-active metal-containingSER-active material; at least one collection component constructed foranalyte sample collection and discharge; at least one containercomponent containing a liquid reagent comprised of a second SER-activemetal-containing SER-active material; and a component for introducingsaid liquid reagent into said SER-active device; at least one of saidfirst and second SER-active materials being functionalized with at leastone binding agent that has the specific capability of binding theretothe at least one designated target analyte, and being accessible for thedeposit thereupon of liquid analyte samples, said SER-active devicebeing constructed for receiving, by way of said component forintroducing, said liquid reagent, and for enabling irradiation, andcollection of Raman scattered radiation cooperatively generated by theSER-active metals of said first and second SER-active materials, by andfrom a Raman spectrometer, all of said components being normallycontained by said packaging means.
 33. The apparatus of claim 32 wheresaid SER-active device component comprises a substrate supporting saidfirst SER-active material.
 34. The apparatus of claim 32 where saidsubstrate is fabricated from metal, glass, paper, or plastic, and is inthe form of a substantially planar sheet, plate, or membrane.
 35. Theapparatus of claim 34 wherein said substantially planar sheet, plate, ormembrane has a multiplicity of wells formed thereinto for receiving ananalyte sample.
 36. The apparatus of claim 33 wherein said substrate isfabricated from glass or plastic and is in the form of a capillary,vial, disc, or lab-on-a-chip channel, said first SER-active materialbeing supported on a surface provided by said substrate.
 37. Theapparatus of claim 32 wherein a linker chemical substance or biochemicalsubstance is interposed for attaching said at least one binding agent tosaid SER-active metal of said at least one of said first and secondSER-active materials.
 38. The apparatus of claim 32 wherein saidSER-active metal of each of said first and second SER-active materialsis selected from the group consisting of copper, gold, silver, nickel,platinum, rhodium, iron, ruthenium, cobalt, nickel, palladium, andalloys and mixtures thereof.
 39. The apparatus of claim 32 wherein saidSER-active metal of said first SER-active material is of particulateform or in the form of a surface having a morphology that isfunctionally equivalent to metal particles for generating a plasmonfield when irradiated, and wherein said SER-active metal of said secondSER-active material is of particulate form.
 40. The apparatus of claim38 wherein said SER-active metals of said first and second SER-activematerials are different from one another.
 41. The apparatus of claim 32wherein said support structure of said SER-active device componentcomprises a chemically synthesized porous structure through which saidliquid reagent comprised of said second SER-active material, said atleast one designated target analyte, and any signature chemicalsubstance that may be present, can readily pass.
 42. The apparatus ofclaim 41 wherein said chemically synthesized porous material iseffective to separate said at least one designated target analyte fromother substances normally contained in an analyte sample.
 43. Theapparatus of claim 32 wherein each of the at least one designated targetanalyte and said at least one binding agent is a chemical, biochemical,or biological substance.
 44. The apparatus of claim 43 wherein said atleast one binding agent and said at least one designated target analyteare paired with one another for effective interbonding, such pairs beingselected from the group consisting of (a) antibodies and antigens, (b)peptides and biologicals, (c) drug receptors and drugs, (d) enzymes andtheir specific biochemical substrates, (e) carbohydrates and lectins,and (f) nucleic acid sequences and their complements.
 45. The apparatusof claim 32 wherein said apparatus comprises a plurality of collectioncomponents, contained by said packaging means, selected from the groupconsisting of eyedroppers, syringes, pipettes, micropipetters, swabs,and various combinations thereof.
 46. The apparatus of claim 32additionally including a water-supply component, contained by saidpackaging means, comprised of a container containing distilled,deionized water.
 47. The apparatus of claim 32 additionally including atleast one mixing vial component contained by said packaging means. 48.The apparatus of claim 32 additionally including a filtering component,contained by said packaging means, for filtering an analyte sample. 49.The apparatus of claim 32 additionally including at least one secondreagent supply component comprised of a container containing a secondreagent, said second reagent being selected from the group consisting ofbuffered solutions, digesting agents, and agents that are reactive todegrade biological materials so as to effect release of signatureanalytes.
 50. The apparatus of claim 49 wherein said second reagent isan agent that is reactive with constituents of analyte samples todegrade biological materials and release at least one signature chemicalsubstance, signature biochemical substance, or signature biologicalsubstance so as to provide a signature analyte constituting the at leastone designated target analyte.
 51. The apparatus of claim 50 whereinsaid agent is selected from the group consisting of acetic acid, adipicacid, ascorbic acid, citric acid, formic acid, fumaric acid, lacticacid, malic acid, palmitic acid, peracetic acid, propionic acid,salicylic acid, sorbic acid, succinic acid, trihaloacetic acid, acetone,acetonitrile, benzene, chloroform, carbon tetrachloride, cyclohexane,dichloromethane, diethyl ether, dimethylsulfoxide, ethyl acetate,ethylene glycol, isopropyl ether, methyl ethyl ketone, n-hexane,phenolic derivatives, tetrahydrofuran, toluene, and mixtures thereof.